One known test procedure or process for detection of a specific bioagent that is applicable to a variety of fields, such as biotechnology, environmental protection and public health, is the enzyme linked immunoassay (hereafter referred to as “ELISA”). The ELISA process constitutes an identification process that uses molecular interactions to uniquely identify target substances. A basic definition of ELISA is a quantitative in vitro test for an antibody or antigen (e.g., a bioagent) in which the test material is adsorbed on a surface and exposed to a complex of an enzyme linked to an antibody specific for the substance being tested for with a positive result indicated by a treatment yielding a color in proportion to the amount of antigen or antibody in the test material. The basic ELISA procedure is described more specifically, for one, in a book entitled Methods in Molecular Biology Vol. 42, John R. Crowther, Humana Press, 1995.
The “antibody specific for the substance being tested for” in the foregoing definition constitutes one type of recognition molecule, a molecule that is capable of binding to either reactant or product molecules in a structure-restricted manner. That is, the recognition molecule binds to a specific three-dimensional structure of a molecule or to a two-dimensional surface that is electrically charged and/or hydrophobic in a specific surface pattern. It may also be recognized that ELISA-like approaches using other recognition molecules can also be used, such as aptamers, DNA, RNA and molecular imprint polymers.
More recently, the foregoing definition for ELISA has been expanded beyond the colormetric approach, in which color and color intensity is used as the reporter or indicia of the antigen or antibody, to include a voltametric or amperiometric approach to detection and assay, in which the rate of change of voltage or current conductivity is proportional to the amount of antigen or antibody contained in the test material. Patent Cooperation Treaty application PCT/US98/16714, filed Aug. 12, 1998 (International Publication No. WO 99/07870), entitled “Electrochemical Reporter System for Detecting Analytical Immunoassay and Molecular Biology Procedures” (hereafter the “16714 PCT application”), claiming priority of U.S. patent application Ser. Nos. 09/105,538 and 09/105,539”), to which the reader may refer, describes both a colormetric and an electrochemical reporter system for detecting and quantifying enzymes and other bioagents in analytical and clinical applications. The electrochemical reporter system of the 16714 PCT application employs a sensor for detecting voltametric and/or amperiometric signals that are produced in proportion to the concentration of organic (or inorganic) reporter molecules by redox (e.g. reduction-oxidation) recycling at the sensor.
In brief, in the ELISA test, the suspect bioagent is initially placed in a water-based buffer, such as a phosphate buffered saline solution, to form a sample solution. That sample solution is mixed with a quantity of particles, beads, the surface of which is coated with a recognition molecule for the suspect bioagent (also sometimes referred to as a receptor molecule). The particular recognition molecule used to coat the beads is known to bind to the bioagent of interest and is a primary recognition molecule (antibody) or “1° Ab.” That is, the recognition molecule coating exhibits a chemical “stickiness” that is selective to specific bioagents.
Any bioagent that is present in the sample solution binds with a non-covalent bond to a respective recognition molecule and thereby becomes attached to a respective one of the beads in the mixture-solution. If the sample solution does not contain a bioagent or if the bioagent that is present in the solution is not one that binds to the selected recognition molecule, then the recognition molecule remains unbound. Further processing of the ELISA process then shows nothing.
Assuming the suspect bioagent is present in the sample, the bioagent bonds to the coating on the beads. The solution then contains a quantity of bioagent molecules bound respectively to a quantity of coated beads. The mixture is optionally washed, as example, in a phosphate-buffered saline, and a second recognition molecule, more specifically, a recognition molecule and enzyme linked combination, is then added to the mixture. The second recognition is also one that is known to bind to the suspect bioagent. The second recognition molecule may either be one that is monoclonal, e.g. one that binds to only one specific molecule, or polyclonal, e.g. a mixture of different antibodies each of which shares the characteristic of bonding to the target bioagent. The enzyme is covalently bound to the second recognition molecule and forms a complex that is referred to as a secondary recognition molecule-enzyme conjugate or “2° Ab-enz.” As known by those skilled in the art, an enzyme is a “molecular scissors”, a protein that catalyzes a biological reaction, a reaction that does not occur appreciably in the absence of the enzyme. The enzyme is selected to allow the subsequent production of an electrochemically active reporter.
The 2° Ab-enz binds to the exposed surface of the immobilized bioagent to form a “recognition molecule sandwich” with the bioagent forming the middle layer of that sandwich. The recognition molecule sandwich coated beads are washed again to wash away any excess 2° Ab-enz in the solution that remains unbound.
The beads and the attached recognition molecule sandwich, the 1° Ab/bioagent/2° Ab-enz complex, in the solution are placed over the exposed surface of the redox recycling sensor. The substrate of the foregoing enzyme is added to the solution and the substrate is cleaved by the enzyme to produce an electrochemically active reporter. The substrate of the enzyme, referred to as PAP-GP, is any substance that reacts with an enzyme to modify the substrate. The effect of the enzyme is to separate, cut, the PAP, a para-amino phenol, the electrochemically active reporter, from the GP, an electrochemically inactive substance.
The foregoing chemical reaction is concentrated at the surface of the sensor. The rate of production of the foregoing reporter (PAP) is proportional to the initial concentration of bioagent. The reporter reacts at the surface of the sensor, producing an electrical current through the sensor that varies with time and is proportional to the concentration of the bioagent, referred to as redox recycling. The occurrence of the electric current constitutes a positive indication of the presence of the suspect bioagent in the sample. Analysis of the electric currents produced over an interval of time and comparison of the values of that electric current with existing laboratory standards of known bioagents allows quantification of the concentration of bioagent present in the initial sample.
Recognizing that need, the present inventors, together with other co-inventors, created an automated test procedure and apparatus, which is described in U.S. patent application Ser. No. 09/837,946, filed Apr. 19, 2001, entitled Automated Computer Controlled Reporter Device for Conducting Immunoassay and Molecular Biology Procedures (hereafter the “946 application”), assigned to the assignee of the present application, the content of which is incorporated herein by reference. The apparatus of the '946 application, hereafter referred to as the automated ELISA system, provides a user friendly stand-alone portable automated system that is able to automatically perform an ELISA test. The automated ELISA system contains a number of solutions in respective reservoirs and pumps that are controlled by a programmed computer. That automated unit may be operated by persons who are not biologists and who require minimal training to operate the unit.
The foregoing automated ELISA system also employs coated beads of magnetic material and a magnetic positioning device to manipulate and position the coated magnetic beads under control of the computer, such as during the washing steps of the ELISA process, and in positioning the beads at the sensor during redox recycling. The automated ELISA system of the '946 application provides a solution that permits wide dispersal of testing units among the general population that takes into account the lesser skills of the prospective operators for those units.
In a first step of the assay procedure the sample solution, containing the sample that is to be tested for the presence of a specific bioagent, is placed in a container (or equivalent vessel) containing the 1° recognition molecule coated magnetic beads, and the contents of the container is mixed together to ensure that the respective parts, that is, molecules, of the sample contacts the coating of a respective bead. If the sample is of the specific bioagent, then the respective parts of the sample link or, as variously termed, stick to the recognition molecule coating of a respective bead. In the automated ELISA apparatus of the '946 application, the sample solution and the coated beads are pumped into the common container by electrical pumps controlled by a programmed computer. Although the injection of the two ingredients into a single container was originally thought to sufficiently mix the two ingredients, current thinking is that mixing with greater thoroughness provides a more accurate assay and a better result.
One result of the process is to determine how much of the bioagent is in the solution; that is, the concentration. The purpose of mixing the solution of sample and coating beads is to ensure that every portion of the sample has an opportunity to strike the coating of a bead in order to attach or link as much of the sample as possible. In practice, the volume of the sample and bead solution is small and the container, which may be a length of pipette tubing, is also small. A known practical way to mix the ingredients of the solution is to create turbulence by repeatedly pumping the foregoing solution out of the container and then pumping that solution back into the container or to recirculate that solution. It is found that the foregoing approach to mixing is not as efficient as desired. Not only must additional pumps and/or valves and plumbing be included in the automated system or the pumps and/or valves that are included in the automated system must be adapted to that task, which increases the complexity of that system, but, more importantly, due to the inefficiency of that mixing process, considerable mixing time is required to ensure that molecules of the sample have the opportunity to link to an available coated bead, an effect here described as thorough mixing.
Accordingly, a principal purpose of the present invention is to bring the molecules of the sample into close association with the recognition molecule coated magnetic beads in the automated system in a shorter period of time and with greater efficiency than previously carried out.
An additional purpose of the invention is to increase the effectiveness of the automated computer controlled reporter device for conducting immunoassay and molecular biology procedures disclosed in the '946 application.